The present invention relates to radiolabeled compounds and their use in radioimaging and/or radiotherapy. More particularly, the present invention relates to radiolabeled irreversible inhibitors of epidermal growth factor receptor tyrosine kinase (EGFR-TK) and their use as biomarkers for medicinal radioimaging such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), and as radiopharmaceuticals for radiotherapy.
The use of radioactive nuclides for medicinal purposes is well known in the art. Biologically active compounds that bind to specific cell surface receptors or that in other ways modify cellular functions have received some consideration as radiopharmaceuticals, and therefore, when labeled with a radioactive nuclide, such compounds are used as biospecific agents in radioimaging and radiotherapy.
Positron Emission Tomography (PET), a nuclear medicine imagine technology which allows the three-dimensional, quantitative determination of the distribution of radioactivity within the human body, is becoming an increasingly important tool for the measurement of physiological, biochemical, and pharmacological function at a molecular level, both in healthy and pathological states. PET requires the administration to a subject of a molecule labeled with a positron-emitting nuclide (radiotracer) such as 15O, 13N, 11C and 18F, which have half-lives of 2, 10, 20, and 110 minutes, respectively.
Single Photon Emission Computed Tomography (SPECT) is a form of chemical imaging in which emissions from radioactive compounds, labeled with gamma-emitting radionuclides, are used to create cross-sectional images of radioactivity distribution in vivo. SPECT requires the administration to a subject of a molecule labeled with a gamma-emitting nuclide such as 99mTc, 67Ga, 111In and 123I.
Polypeptides such as growth factors, differentiation factors, and hormones often mediate their pleiotropic actions by binding to and activating cell surface receptors with an intrinsic intracellular protein tyrosine kinase activity. Epidermal growth factor receptor-tyrosine kinase (EGFR-TK) is over is expressed in breast cancer and other neoplasia. A suitable radiotracer that binds to EGFR-TK might allow, through a nuclear medicine imaging technique such as PET and SPECT, the mapping and quantification of this receptor-kinase. This would allow the study of changes in levels of expression of this receptor, including the monitoring of response to hormonal or other chemotherapy, and could lead to better patient management and differentiation in regard to therapeutic course of action.
Moreover, such radiotracer that comprises a suitable radioactive nuclide can be further used as an EGFR-TK biospecific agent for radiotherapy.
Recently, 99mTc-labeled anti EGFR antibody was synthesized and biodistribution and dosimetry studies were performed in humans [1, 2]. However this labeled antibody, similar to other protein radiopharmaceuticals, has high and prolonged retention of radioactivity in the liver which constitutes a major problem for clinical applications. Furthermore, the researchers found that it was difficult to obtain accurate quantification of activity in tumors within normal organs because of varying background activities, particularly in lung lesions where fluid and atelectasis could not be differentiated from tumor.
EGF itself has been labeled for nuclear medicine imaging with gamma emitting nuclides including 99mTc [3, 4] and indium-111 [5, 6), and the positron-emitting nuclide bromine-76 [7, 8]. The biodistribution in normal rats of the latter, bromine-76 EGF (murine), was reported [8], but no other in vivo studies in laboratory animals or humans have been reported.
4-Anilinoquinazolines, also referred to herein as 4-(phenylamino)quinazolines, have been shown to potently and selectively inhibit EGFR-TK activity by binding reversibly to an inner membrane ATP binding site on EGFR-TK, the prototype for such compounds being the small-molecules PD 153035 [9] and AG 1478 [10]. A report of a radioiodinated analog of PD 153035 including in vitro binding studies in MDA-486 cells has been presented [11].PD 153035 labeled with carbon-11 in the 6,7-methoxy groups has been evaluated in rats implanted with human neuroblastoma xenografts (SH-SY5Y) but specific uptake was not determined in a blocking study [12]. PD 153035 was also labeled with carbon-11 specifically in the 7-methoxy position and biodistribution experiments were performed in normal mice, but uptake specificity could not be demonstrated as administration of an enzyme-blocking dose of PD 153035 caused an increase in tracer uptake in the tissues studied [13]. The same abstract reported the labeling of the 7-(2-fluoroethoxy) PD 153035 analog with fluorine-18, but no biological experiments with this tracer were described. Additionally, the 2-18F-fluoroethyl group might be subject to a high rate of 18F-hydrogen fluoride elimination to give the corresponding alkene ether, potentially resulting in high uptake of fluorine-18 in bone, giving poor in vivo images. Further, these ultra potent (IC50<30 pM) inhibitors may only measure flow or permeability surface area rather than biochemical changes [14].
U.S. Pat. No. 6,126,917 teaches 4-(anilino)quinazoline derivatives, reversible inhibitors of EGFR-TK, labeled with fluorine-18 on the aniline ring. These compounds were tested in vitro, in vivo and by PET image analysis. While some of these compounds showed effective (reversible) inhibition activity in vitro, they were found to be ineffective as tracers for the imaging of EGFR-TK in vivo due to kinetic factors such as kon and koff and rapid blood clearance, as was fiber demonstrated by an animal PET comparative study between fluorine-18 FDG and these radiolabeled compounds. It is assumed that the discrepancy between the encouraging in vitro results and the discouraging in vivo results derives from the ATP competition at the compounds' binding site.
Thus, in order to achieve better imaging results, the non-specific binding of the radiolabeled compounds should be reduced. This can potentially be achieved by the use of derivatives of irreversible EGFR-TK inhibitors that are labeled with a positron-emitting nuclide. The irreversible binding of such compounds could potentially result in higher diagnostic performance. Furthermore, such irreversible inhibitors, when labeled with a suitable radioactive nuclide, can be used as effective radiotherapy agents as well, based on their high affinity toward, and irreversible binding to, tumor cells expressing EGFR-TK. Thus, such radiolabeled compounds that are targeted to the EGF receptor can bind preferentially to tumor cells and would lead to a high effective concentration of the radionuclides and therefore cause preferential cell killing at the site of the tumor.
Irreversible EGFR-TK inhibitors were recently described [15, 16, 19 and U.S. Pat. Nos. 6,153,617 and 6,127,374]. The irreversible binding thereof is achieved by 4-(anilino)quinazoline derivatives that are substituted at the 6 or 7 position of the quinazoline ring with an αβ-unsaturated carboxylic group, preferably an acrylamide group, which binds covalently to the Cys-773 at the EGFR-TK ATP binding site. Some of these compounds showed high potency toward EGFR inhibition in both in vitro and in vivo experiments. However, these compounds were not radiolabeled, and therefore cannot be used for radioimaging or radiotherapy.
There is thus a widely recognized need for, and it would be highly advantageous to have, radiolabeled irreversible inhibitors of EGFR-TK for use in radioimaging and radiotherapy.